The action of glutamate-activated ion channels determines the flow of information via excitatory synapses throughout the mammalian brain. As a result, the normal function and regulation of glutamate channels (of the AMPA, kainate and NMDA subtypes) are involved in virtually all brain functions. In the past 10-15 years, fundamental studies of N- methyl-D-aspartate (NMDA) receptors provide one of the clearest rationales for the relevance of basic research to clinical problems. These studies have provided new insights into normal brain functions such as synaptic plasticity, the formation of memories, and the action of psychomimetic drugs such as phencyclidine (PCP) on human behavior. Excessive stimulation of glutamate receptors can cause neuronal cell death in seizures and stroke, and may play an important role other neuropsychiatric illnesses. An amazing complexity of regulatory mechanisms influence glutamate receptors. For example, NMDA receptors are regulated by allosteric mechanisms, multiple kinases, phosphatases and soluble second messengers. Although such complexity may seem fitting given the central role of excitatory synapses, the question of what determines the specificity of such interactions is unexplored. Calcium influx into neurons through open NMDA channels at synapses initiates several of these regulatory mechanisms, thus we have focused on the regulation of hippocampal NMDA receptors by intracellular calcium. Our results suggest that compartmentalization and local interactions between glutamate receptors, regulatory proteins and cytoskeletal elements in the postsynaptic density (PSD) are keys to this puzzle. These interactions are likely to affect the activity of synaptic NMDA channels as well as the formation and receptor composition of hippocampal synapses. We will test two aspects of this general hypothesis in this proposal. First, we will examine the domains of the NMDA receptor responsible for calcium regulation (Aim 1-2) and desensitization (Aim 3). Preliminary results demonstrate that calcium regulation is NR2A specific and chimeric/deletion constructs suggest regions of NR1 and NR2A that are involved, perhaps by a ball-and-chain mechanism. We will also examine the possible inductive role of NMDA receptors, and the NR2B subunit in particular, in the function and localization of individual synapses on hippocampal neurons (Aim 4). These studies will make use of transgenic mice lacking the NR2B subunit. The proposed studies will use recombinant NMDA receptors expressed in 293 cells and Xenopus oocytes as well as native receptors in cultured hippocampal neurons. Novel methods we developed for studies of synaptic NMDA receptors and function of individual synaptic sites will be used.